2. Polyisobutylmethacrylate (see pg. 99) is prepared as a 10% (w/v) solution in
chloroform which is diluted 1/100 into rapidly stirring hexane to generate a 1 mg/ml stock
which can be stored at room temperature. The solution is further diluted immediately
before use with additional hexane to a final concentration of 2-200 µg/ml depending on
the cell type. Developed and dried TLC plates are dipped sequentially for 30 sec. each in
hexane, then in PIBM solution, then allowed to air dry.

3. The PIBM-coated plate is immersed in medium until wet, then transferred into medium
containing blocking agent 0.5 mg/ml BSA) for 30 min., then into medium without blocker.

4. The preblocked plate is placed sorbent side down on the spacers of the adhesion
chamber. Medium is pipetted onto the back of the TLC plate and the rubber gasket applied
such that air bubbles are excluded from the plate-gasket contact area. The gasket and
acrylic cover are fixed in place with the screws supplied.

5. Any medium trapped in the chamber is decanted through the luer lock openings at the
end of the Chamber. Cell suspension (105-106 cells/ml) in medium
containing 1-5 mg/ml BSA is pipetted into one of the openings until the chamber is full
and free of air bubbles (chamber capacity 15 ml). The end openings are sealed with luer
locks, and the sealed box is placed with TLC sorbent side up such that the cells settle
onto the plate surface. The box is placed on a microplate centrifuge carrier and
centrifuged at 30xg, for 3 min. to bring cells into contact with the TLC plate surface.

6. The chamber is immersed (sorbent side up) in a water bath typically at 37°C for 30
min. to allow adhesion to occur.

7. The chamber is removed from the water bath, inverted, placed (sorbent side down) in
a centrifuge carrier, and centrifuged (250-500xg, 10 min., 4°C to remove non-adherent
cells from the TLC plate surface.

8. While still inverted, the chamber is fully immersed in an ice-cold tub of PBS, the
cover and gasket removed, and the TLC plate is gently removed, righted (while immersed)
and placed in the transfer dish. The fluid-filled dish is then used to transfer the TLC
plate through two 150 mm Petri dishes filled with PBS to wash.

9. Still in the transfer dish, the plate is placed in an empty 150 mm Petri dish and
gently overlaid with 100-150 ml of 2% glutaraldehyde in PBS at ambient temperature for 30
min.

10. The TLC plate is removed from the transfer dish, washed by immersion in PBS and
placed vertically to dry.

11. The thoroughly dried plate is placed on X-ray film and exposed. After exposure for
1-24 hr. the film is processed to detect the position of adherent cells.

Note: If glycolipids are present at sufficient concentration (>500
pmol/lane) they may be detected on the TLC plates after autoradiography by incubating the
plate in Coomassie Blue (0.3mg/ml) in methanol: water (1:5) for 30 min. followed by
destaining in the same solvent mixture for 5 min. Coomassie Blue treatment also stains
adherent cells which can be visualized by light-field microscopy at the sites of
autoradiographic bands to confirm that the radiolabel detected corresponds to intact cell
adhesion.